During infection of Escherichia coli, bacteriophage T4 usurps the host transcriptional machinery, redirecting it to the expression of early, middle, and late phage genes. This machinery is driven by E. coli RNA polymerase, which, like all bacterial polymerases, is composed of a core of subunits (beta, beta', alpha1, alpha2, and omega) that has RNA synthesizing activity and a specificity factor (sigma). The sigma protein identifies the start of transcription by recognizing and binding to sequence elements within promoter DNA. During exponential growth, the primary sigma of E. coli is sigma70, which, like all primary sigmas, is composed of four regions. Sigma70 recognizes DNA elements around positions -10 and -35 of host promoter DNA, using residues in its central portion (regions 2 and 3) and C-terminal portion (region 4), respectively. In addition, residues within region 4 must also interact with a structure within core polymerase, called the beta-flap, to position sigma70 region 4 so it can contact the -35 DNA. T4 takes over E. coli RNA polymerase through the action of phage-encoded factors that interact with polymerase and change its specificity for promoter DNA. Early T4 promoters, which have -10 and -35 elements that are similar to those of the host, are recognized by sigma70 regions 2 and 4, respectively. However, although T4 middle promoters have an excellent match to the sigma70 -10 element, they have a phage element (a MotA box) centered at -30 rather than the sigma70 -35 element. Two T4-encoded proteins, a DNA-binding activator (MotA) and a T4-encoded co-activator (AsiA), are required to activate the middle promoters. AsiA alone inhibits transcription from a large class of E. coli promoters by binding to and structurally remodeling sigma70 region 4, preventing its interaction with the -35 element and with the beta-flap. In addition to its inhibitory activity, the AsiA-induced remodeling allows the N-terminal domain of MotA (MotA NTD) to bind to the C-terminus of sigma70 and the C-terminal domain of MotA (MotA CTD) to bind to the MotA box. This process is called sigma appropriation. Despite dozens of activator crystallographic structures and RNA polymerase structures, there is only one complete structure of an activator/RNA polymerase/DNA complex. However, the type of activation performed by this crystallized complex is fundamentally different from that of sigma appropriation. We previously combined biochemical analyses, available structures, and modeling to develop a structural model of sigma appropriation. Our work depicted how AsiA/MotA redirects sigma70, and therefore RNA polymerase activity, to a T4 middle promoter DNA and how the flexibility of sigma70 region 4 is likely crucial for this process. Our work suggested that MotA interacts with its DNA binding motif using a previously unidentified interaction mechanism in which the double wing helix structure of the CTD contacts the major groove of the DNA and the linker contacts the minor groove. In collaboration with the laboratory of Dr. Steve White (St Judes), we solved the crystal structure of the MotA linker-CTD with the DNA, revealing a new mode of protein-DNA interaction. The CTD domain binds DNA mostly via interactions with the DNA backbone, but the binding is enhanced in the specific cognate structure by additional interactions with the MotA box motif in both the major and minor grooves. The linker connecting the two MotA domains plays a key role in stabilizing the complex via minor groove interactions. The structure is consistent with our previous model derived from chemical cleavage experiments using the entire transcription complex. Alpha- and beta-D-glucosyl-5-hydroxymethyl-deoxycytosine replace cytosine in T4 DNA, and docking simulations indicate that a cavity in the cognate structure can accommodate the modified cytosine. Our binding studies have confirmed that the modification significantly enhances the binding affinity of MotA for the DNA. Our work reveals how a DNA modification can extend the uniqueness of small DNA motifs to facilitate the specificity of protein-DNA interactions. E. coli host promoters use specific regulators to control initiation, elongation, and termination. While activators and repressors typically interact with specific DNA sequence motifs, DksA is a member of a growing class of global transcription factors, called secondary (2) channel proteins, that interact with just RNA polymerase. The interaction of DksA with RNA polymerase generates one of two binding sites for the small molecule ppGpp on polymerase, and in most cases, DksA activity is dependent on ppGpp. We have investigated the effects of a DksA or ppGpp deletion on T4 growth and transcription using primer extension, RNA-seq, RT-qPCR, single bursts, and a semi-automated method to document plaque size. We demonstrated that a deletion of either DksA or ppGpp results in significantly larger plaques for either T4 wild type (wt) or a T4 motA knockdown (T4motAam). Infections in a ppGpp0 host have a marginal effect on burst size, latent period, or phage transcript abundance. However, a deletion of DksA results in a 2-fold increase in T4 wt burst size and increased levels of several early promoter RNAs. As the T4 activator MotA is required for T4 middle promoter activation, a T4 motA knockdown results in poor phage growth. We have provided the first global transcriptome analyses of T4 wt and T4motAam infections of a wild-type laboratory strain and a dksA mutant host strain. The transcriptome of T4motAam revealed that middle genes are affected differentially, ranging from slight to severe inhibition of gene expression. We show that the absence of DksA in the T4 motA knockdown background also increases transcription from specific phage early promoters. This increase ameliorates the poor growth of T4motAam by increasing expression of downstream middle genes needed for replication, recombination, and late transcription. As we do not observe an effect of DksA on early promoter transcription in a purified in vitro transcription system, we conclude that DksA decreases T4 early transcription through other changes present in the infected host or by using additional host/phage factors. Viruses have evolved multiple means for overcoming defense mechanisms in their host to allow for optimal viral proliferation. In bacteria, histone-like proteins, such as H-NS and StpA, are DNA binding proteins that form higher-order nucleoprotein complexes that typically repress transcription by targeting AT-rich DNA sequences. As phage genomes often display a high AT content, H-NS can protect bacteria from the expression of phage-encoded genes by preferentially binding these sequences. Bacteriophage T4 has a much high %AT content than its host E. coli, 65.5% AT vs. 45% AT, respectively, suggesting that H-NS might be deleterious to T4 growth. We have found that despite being nonessential, the T4 gene motB is conserved among T4-type phages and that a motB amber mutant (T4motBam) produces a two-fold lower burst size compared to T4 wild-type infections. We discovered that MotB production is extremely toxic when expressed in E. coli resulting in the decondensation of host DNA, cell lengthening, significant reduction in actively dividing cells compared to a vector control, and cell lysis. MotB binds tightly and nonspecifically to both host and T4 DNA and co-purifies with bacterial H-NS and StpA. Footprinting assays revealed the MotB, like H-NS, spreads along large regions of DNA. Our results indicate that the T4 motB gene encodes a bactericidal DNA binding protein that improves the fitness of T4 infections. We hypothesize that the interaction of MotB with DNA may be part of a mechanism used by T4 to disrupt H-NS dependent DNA condensation, leading to a more productive infection.